Effective gene therapy for hereditary neurologic disease will require the development of safe vectors, which can confer stable transgene expression on neurons. As a model disease we have chosen ataxia-telangiectasia (A-T), an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition and radiation sensitivity. These studies will focus on the development of plasmid-based HSV amplicon vectors to achieve site-directed integration of transgenes into the cell genome or to correct mutations in endogenous genes by homologous recombination (HR). Replacement vectors will incorporate elements of AAV, including ITRs and Rep, to target insertion of the ATM cDNA into an AAVS1 site engineered into the genome of Atm knock-out mice. Vectors for HR will include 8 and16 kb regions of the normal mouse gene spanning a 9 bp deletion in the Atm gene in knock-in mice, as well as sequences and expression cassettes for elements that should increase the frequency of HR, such as Rad51 and HSV ICP8. Successful integrations will be evaluated in cultured fibroblasts and neuroprecursor cells from these mice, as well as in primary neuronal cultures and in neurons in the cerebella of these mice by expression of the transgene (immunocytochemistry and RT-PCR) and analysis of recombinant events (PCR, Southern blots and sequencing). Functional recovery will be assessed following injection of vectors in the newborn mouse cerebellum by restoration of the normal apoptotic response of neurons to ionizing radiation, as well as by long-term improvement in motor function, reduction of oxidative damage and expression of ATM. These therapeutic vector designs have applications for a broad range of neurologic diseases allowing delivery and stable expression of large transgene cassettes and the potential for permanent correction of gene defects in neurons.